Exotoxin A and the siderophores (pyochelin and pyoverdine) are produced in P. aeruginosa by a complicated process that involves several genes. The long-term goal of this proposal is to define the mechanism that regulates the synthesis of exotoxin A and pyoverdine in P. aeruginosa and to identify the different factors that participate in this regulation. We have identified a gene, ptxR, which positively regulates the transcription of toxA, the toxA regulator regA, and the genes that code for some components of the pyoverdine system. Available evidence suggests that the regulation of these genes by ptxR is indirect (through an independent gene). In addition, the regulation of ptxR expression itself appears to involve several regulatory proteins, both identified and as yet unidentified. One identified protein, the alternative sigma factor PvdS, also regulates toxA and the pyoverdine genes. The unidentified proteins bind specifically to the ptxR upstream region. The specific aims of this proposal are: 1) to determine the mechanism through which toxA and the pyoverdine genes are regulated by ptxR. This includes identification of the ptxR target sequence and the gene that carries it and characterization of the genes that are regulated (directly or indirectly) by ptxR; 2) to examine the regulation of ptxR expression in P. aeruginosa. This includes extensive in vivo and in vitro transcriptional analysis of the ptxR upstream region and examining ptxR expression in different specific P. aeruginosa mutants; and 3) to isolate and characterize the putative regulatory proteins and to identify the nucleotide sequences to which they bind. These aims will be accomplished using protein purification experiments, simple and modified DNA/protein binding experiments, functional genomics, 2-D gel analysis, in vivo and in vitro transcription, and different recombinant DNA techniques.